‘w —' ‘-—-_—N—— __ . , . . . . . . . I ‘ ' V a. . .1 - ..I l r I o .'.I l‘. . ‘ . ,... . I,‘\"°" ".- 'l ' ~ " ' . M s. u ¢I. .o III . l- . ‘ "‘ ‘ ’ ' . ,0. '.|I‘ uvt-l-o . .*—v-- -M—v.-QV.’ -9 u—o—a—o o—o-‘o -._-. OO—Q'Fn-A- ?”O—--....-----~“.-.“«W BOVINE SERUM PROTEIN. HORMONE CONCENTRATION DURING-LATE . PREGNANCY. PARTURITION AND EARLY LAC'TATION. Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY. WINSTON INGALLS 1972 — ’— -——_-—-—-—_ n i. — > _ _ . . . - a . u- .Wm. - Elm! .m. / LIBRARY Michigan State University ? an: 6‘??? 7 HUAG 8 SONS’ BOOK BINDERY INC. LIBRARY BINDERS g::.1:rcar,mcum_u ABSTRACT BOVINE SERUM PROTEIN HORMONE CONCENTRATION DURING LATE PREGNANCY, PARTURITION AND EARLY LACTATION BY Winston Ingalls Jugular serum was collected from 32 heifers daily from 6 days before to 5 days after parturition, otherwise twice weekly from 30 days before parturition, until first estrus. Serum prolactin ranged from 50 to 100 ng/ml until 2 days before parturition, exceeded 200 ng/ml during the 2 days before parturition and declined to about 60 ng/ml by 60 hours postpartum. The values ranged between 50 and 100 ng/ml throughout the remainder of the postpartum per- iod. Serum growth hormone varied from 4 to 7 ng/ml before parturition, increased to 12 ng/ml for about 36 hours be- ginning at parturition and then decreased to prepartum levels. Luteinizing hormone in blood serum did not change measurably from 30 days before parturition until 4 days postpartum when a gradual increase commenced. A peak of 1.7 ng/ml was noted on day 12 after calving. Changes in release of prolactin, growth hormone, and LH are asynchro- nous around parturition. BOVINE SERUM PROTEIN HORMONE CONCENTRATION DURING LATE PREGNANCY, PARTURITION AND EARLY LACTATION BY Winston Ingalls A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy Science 1972 ACKNOWLEDGMENTS The author wishes to express his gratitude to Dr. Charles Lassiter of the Dairy Science Department who provided facilities and financial assistance for his graduate studies. His sincere appreciation is extended to Drs. E. M. Convey, H. D. Hafs and L. A. Edgerton for their advice and guidance throughout his graduate studies and in the prepa- ration of this thesis. To his student colleagues who assisted in all aSpects of the research being reported he expresses his sincerest appreciation and thanks. Finally he is grateful to Mrs. Valdin Smith for the typing of the bulk of this thesis manuscript. ii TABLE OF CONTENTS LIST OF TABLES . . . LIST OF FIGURES . . INTRODUCTION . . . REVIEW OF LITERATURE . Gestation Period . Factors Affecting Parturition . . . Length of Gestation Non-Endocrine Factors Affecting Parturition . Uterine Contractility Near Parturition . . . . . . . Phases of Parturition . . . . Endocrine Pattern Near Parturition . Estrogens . . Progesterone . Adrenal Corticoids Prolactin . . Growth Hormone . Luteinizing Hormone . . . . . Postpartum Period . Reproductive Tract Changes . . . Ovarian Activity Postpartum Estrus Activity . . . Endocrine Pattern iii Page vi 1O 12 13 15 17 18 18 19 19 21 MATERIALS AND METHODS . . . . . EXperimental Design . . . . Estrus Detection . . . . Blood Handling Procedure . . Radioimmunoassays . . . . General Principles of Radioimmunoassay Antibodies . . . . . . Labeled Hormone Preparation . Radioimmunoassay . . . . RESULTS 0 O . . O O O O 0 Pre and Postpartum Serum Prolactin, GH LH Concentration in Heifers . Serum Prolactin . . . . . Growth Hormone . . . . . Serum LH . . . . . . . DISCUSSION 0 0 O O O O O 0 SUMMARY AND CONCLUSION . . . . BIBLIOGRAPHY . . . . . . . . APPENDICES Appendix I. Composition of Reagents Used in Radioimmunoassay . . II. Mean LH Values Based on 32 Observations Per Day . . . iv Page 22 22 22 23 23 23 24 25 26 29 29 29 32 35 37 42 44 52 56 LIST OF TABLES Table Page 1. Radioimmunoassay Procedure . . . . . . . 28 Figure 1. LIST OF FIGURES Page Radioactivity elution profile for Bio-Gel P-60 chromatography of iodinated luteinizing hormone (LH). The first peak represents iodinated LH and the second peak represents free iodine . . . 27 Serum Prolactin from 26 days prepartum through 26 days postpartum . . . . . 30 Serum Prolactin from 9 days postpartum through 9 days postpartum . . . . . . 31 Serum growth hormone from 26 days post- partum through 26 days postpartum . . . 33 Serum growth hormone from 9 days pre— partum through 9 days postpartum . . . 34 Serum luteinizing hormone from 26 days prepartum through 26 days postpartum . . 36 vi F H awn+¢ any»; 1 h; a.““ meats H rare: INTRODUCTION Knowledge of endocrine events that control concep- tion, pregnancy and lactation could be of fundamental eco- nomic importance to the animal industry. Understanding endocrine involvement in these processes could provide the basis for therapeutic manipulation to Optimize reproductive efficiency and lactation. In addition, understanding repro- duction and lactation disorders requires data from normal animals for comparison. Reproductive efficiency can be increased by shorten- ing the interval from parturition to conception. Improve— ments in nutrition, health management and estrus detection have all been shown to reduce this interval. But in many cases it would appear that endocrine imbalance may cause a prolonged calving interval. Knowledge of normal endocrine parameters during the early post-partum period would allow us to appraise and possibly correct endocrine disfunction. The obvious goal of research dealing with initiation of lactation is to bypass the necessity of pregnancy by using hormone therapy to induce udder growth and cause the onset of copious milk secretion. Attempts have been made e be "Pf": b'J ..‘.4.1.-. :Lent i . ”yr-e. F dew" x... . I Fn- n. o‘Ju-V:‘ I “3""!7 ' H‘au'vv ‘ . I ‘\ Partd: §‘p VA to hormonally establish lactation but variability of re- sponse coupled with lesser milk yields than during a normal lactation has limited its use. Perhaps when we gain suffi— cient information concerning endocrine control of mammary growth and lactation we shall be able to simulate the bio- logical changes that occur in the mammary gland during preg- nancy and parturition and artificially produce lactation. This thesis research was undertaken to define, in part, endocrine changes occurring during late pregnancy, parturition and early lactation in cattle. It is hoped that the changes in serum growth hormone, prolactin and lutein— izing hormone concentrations described here-in will add to a growing body of knowledge which will eventually allow optimum control of reproductive and lactational performance. 32‘ awls ‘ n‘ N \ :If‘ 600‘ V4 wide REVI EW OF LI TE RATURE Gestation Period The time interval between fertilization of an ovum and expulsion of a fully developed fetus is referred to as the gestation period, or gestation. Its duration is in— fluenced by many factors and drastic alteration in length may be detrimental to both fetus and dam. The term partur- ition refers to the physical act of expelling the fetus. Factors Affecting Length of Gestation For most dairy cattle breeds the gestation period is 278—284 days (Salisbury and VanDemark, 1961). In Brown Swiss cattle it is 290 days. Herman and Spalding (1947) stated that gestation length was affected by breed differ- ences. They also reported that: (1) Multiple fetuses re- duced gestation length by approximately 8 days; (2) the gestation period was slightly shorter for heifers than for older cows; (3) cows calving in the fall and winter carried their young one to three days longer than animals calving during the spring and summer; and (4) male calves were car- ried one day longer than female calves. Cases of abnormal length gestation are of interest when searching for clues as to what controls timing of par- turition. Mead 2E 31. (1949) observed prolonged gestation periods in cattle (310-350 days) and reported that most of the calves delivered were hypoglycemic and failed to sur— vive more than a few hours. Subsequent, pedigree examina- tion revealed the syndrome to be elicited by a single auto- somal recessive gene and the dam of any fetus homozygous for this gene exhibited prolonged gestation. In addition, the fetal pituitary was absent and fetal adrenals were hypoplastic. This disorder provided evidence that a normal functioning fetal endocrine system is needed in cattle to insure a normal length gestation. A condition displaying the same trend and a similar genetic pattern was described by Stormont 33 El. (1956) wherein the fetus failed to grow after seven months and gestation was extended up to 500 days. Ewes ingesting skunk cabbage (Veratum Californicum) produced lambs with cephalic abnormalities and displayed prolonged gestation (Binns SE 21., 1963). Similarly, feed- ing sheep the shrub Salsola tuberculata during the final 50 days of gestation resulted in lambs with small pitui- taries and hypOplastic adrenals (Basson EE.E£" 1969). These cases of prolonged gestation involve interference with a component of the fetal pituitary—hypothalmic- adrenal axis which apparently is essential for a normal duration of gestation in sheep and cattle. Shortened rather than prolonged gestation is more common in farm animals. Bacterial agents are the most fre- quent cause of premature deliveries. Osburn 32 31. (1969) were able to induce abortions within a few days following intrauterine inoculation of pregnant cows with vibrio fetus. Plasma progesterone levels always drOpped near the time of fetal expulsion but the timing of the progesterone decline was dependent on the stage of gestation. Abortions during the latter stage of pregnancy (7-8 months) followed 24 hours after a rapid decline in blood progesterone similar to the normal decline in maternal prOgesterone changes pre- ceding normal births. The examples cited demonstrate the importance of both the fetal and maternal endocrine systems for normal gestation and parturition in sheep and cattle. Parturition Non—Endocrine Factors Affecting Parturition The size and weight of the near term fetus may cause uterine distension and irritability which in turn could cause increased uterine contractions and initiation of labor (McDonald, 1969). Severe uterine distension near parturition may cause decreased uterine blood flow result- ing in waste product build up which may trigger parturi- tion. Baird and McDonald (1964) reported a gradual and consistent decrease in oxygen consumption by the maturing v!‘ no' UV" " , . -alu 4 . 5? Lnoe In: lite bovine placenta from the fifth month until term, perhaps indicating placental aging which eventually may signal the onset of parturition. Uterine Contractility Near Parturition Gillette and Holm (1963) studied uterine and ab— dominal wall contractions in cattle by means of balloon implants attached to strain gauges. Uterine contractions 2-4 days prepartum were irregular, brief and uncoordinated. In contrast, uterine contractions were regular, prOpaga- tive and longer in duration beginning 2 hrs before and continuing through parturition. In addition, abdominal muscle contractions during parturition were strong, rapid and appeared to commence with the movement of the allantoic sac through the cervix reaching a peak 10 minutes pre- partum. Despite the strength of the abdominal contractions these workers considered uterine contractions to accom- plish 90 percent of the work of fetal prOpulsion at par- turition. Postpartum uterine contractions were expulsive and rapid for up to 24 hours. Hindson EE.E£' (1965) found no changes in intra- uterine pressure in ewes due to myometrial contractions until 12 hours before fetal delivery and even then no con- sistent pattern was observed. Cervical dilation occurred most rapidly during the last hour prepartum and this ap- peared to initiate intense abdominal muscle contractions which corresponded with the peak of uterine pressure waves. Immediate postpartum contractions were similar to those of the prepartum period. Stimuli for uterine contractions are maximum near parturition but coordinated contractions will not result unless the uterus is reSponsive. Csapo (1950), working with rats and rabbits, reported that as pregnancy progressed uterine actomyosin content increases thereby increasing contractile capacity. Despite this increase in potential contractility the uterus remains quiescent until term. Csapo (1956) theorized that during most of pregnancy pro- gesterone prevents the uterus from contracting when stimu- lated. He found that electrical stimulation of the uterus of early pregnancy resulted in local, nonprOpagated contrac- tions whereas similar stimuli applied to the uterus at term or early postpartum produced propagated contractions which moved over its entire surface. Based on these observations, Csapo and Woodbury (1963) suggested that somehow the inter- cellular conduction process is facilitated near parturition. Phases of Parturition Parturition or fetal expulsion has been separated into three phases by Marshall (1952) and reviewed by Salis— bury and Vandemark (1961) and McDonald (1969). During stage one gradually strengthening myometrial contractions begin and cervical dilation commences. The fluid filled fetal membranes are forced into the cervix along with the forelimbs of the fetus resulting in even more strenuous uterine contractions. During this prepara~ tory stage in monotocous species, the fetus which normally lays on its back is rotated into a dorsal position with its head and forelimbs forcing against the cervix. In cattle this stage is generally limited to a few hours. The second or expulsive stage results in complete cervical dilation and extremely strong uterine and abdominal muscle contractions, which force the fetus through the birth canal. In cattle this is generally accomplished in approxi- mately 20 minutes but may last two hours, especially in first calf heifers. Following fetal delivery continuous, strong uterine contractions persist. During this third phase fetal mem- branes, blood and other fluids are expelled. If the fetal membranes have not been delivered by 12 hours in the cow, it is generally considered pathological and referred to as a "retained placenta." Postpartum uterine contractions aid in reducing uterine size and constricting blood vessels to prevent excess hemorrhaging. Endocrine Pattern Near Parturition Many factors are involved in parturition and some of the possible control mechanism have been alluded to in the previous discussion. Despite all of these contributing forces, control of parturition is probably dominated by the endocrine system. Estroqens Levin (1945) demonstrated increased concentrations of estrogens in the feces and urine of cattle during the final two weeks of pregnancy. Similar results have been reported for laboratory animals (Meites and Turner, 1948). Hunter gt 31. (1970) reported that urinary excretion of total estroqens and estradiol l7-a increased continually during the final 30 days prepartum and peaked either at parturition or 12 hours after. Timing of the estrogen peak was primarily dependent on the length of gestation. Estrone and estradiol 17-8 were unchanged during the last month of pregnancy. Breed, twining and stage of gestation significantly affected prepartum urinary excretion rates. The greatest increase in estrogen excretion occurred during the final 40 hours prepartum, but estrogen excretion de- clined continuously up to 48-72 hours postpartum (Mellin 33 31., 1966). Estradiol 17-8 in the urine continued to increase until 16 hours postpartum and declined thereafter. Holm and Galligan (1966) observed a marked and steady increase in plasma estrone and total estradiol con- centration during the final month of normal pregnancy, followed by a sharp postpartum decline. In contrast, cows exhibiting prolonged gestations showed elevated urinary estrogens 25-40 days before eXpected calving and a steady decline thereafter with no increase at expected term. The significance of this observation is unknown, but it does 10 suggest that proper serum estroqen concentration may be required to insure normal parturition. Urinary estrogen concentration, although recognized to be of limited value as an estimator of ovarian function, does indicate a trend toward increasing estroqen secretion during the last month of pregnancy. Recent develOpment of radioimmunoassays capable of detecting plasma levels of estroqens should provide more sensitive estimates of altered estroqen secretion. Progesterone Progesterone secretion patterns during pregnancy have been well documented for many species. In the bovine, plasma progesterone concentration fluctuates a great deal during the course of gestation (Randel and Erb, 1971). Erb 25 al. (1968a) demonstrated no significant change in corpus luteum content and concentration of progesterone during pregnancy. Short (1960) reported that blood progesterone concentration in cattle was elevated until the last month of pregnancy declining thereafter until parturition” ‘Hunter 22 El. (1970) confirmed this gradual decline in progester- one during this final month. POpe gt'al. (1969) and Stabenfeldt gt El. (1970) indicated a more rapid decrease in progesterone concentration about 24 hours prepartum in cattle. Smith SE 31. (1971) indicate that the rapid pre- partum progesterone decline may commence as early as 3.5 days prepartum in Holstein heifers. 11 Although the corpus luteum of pregnancy is not re- quired in several species (Hafez, 1968) during the third trimester of pregnancy, most studies indicate that gesta- tion is not normal following ovariectomy at 200 days ges- tation in cattle (Estergreen 35 31., 1967). Generally if exogenous proqesterone is not administered gestation is shortened, calving difficulties occur and fetal membranes are nearly always retained (Tanabe, 1970). Evidently extraovarian sources of progesterone are insufficient to support normal gestation and parturition. Using in vitro methods, Ainsworth and Ryan (1967) demonstrated the ability of placenta from late pregnant cattle and sheep to convert pregnenolone-7-3H to radio- active progesterone.' Many metabolites of progesterone were also recovered with the pregnane derivatives pre- dominating. These results indicate a placental capacity to both synthesize and metabolize progesterone. The low blood progesterone during late pregnancy may only be necessary for proper parturition and decidua- tion of the placenta. Why ovarian progesterone production decreases as parturition approaches is unknown. Mills and Morrissette (1970) demonstrated that progesterone syntheses by ovaries from cattle during early and late pregnancy re— sponded equally well to luteinizing hormone stimulation. They postulated that decreased progesterone secretion during late pregnancy may be due to a decreased 12 concentration of circulating gonadotropin. This is doubt- ful in light of data reported by Randel and Erb (1971) which demonstrated no change in plasma LH from day 7 to day 260 of pregnancy. Adrenal Corticoids Glucocorticoids are steroid hormones secreted by the adrenal cortex which are necessary for normal parturi- tion in sheep and cattle. Adams and Wagner (1970) observed a significant in- crease in bovine plasma corticoid concentrations to a plateau at 4 days prepartum which continued through normal parturition and declined thereafter. Smith gt 21. (1971) found that serum glucocorticoid concentration increased 12 hours prepartum, remained increased at parturition and declined to prepartum levels by 12 hours after calving. Inactivation of cortisol by protein binding in serum is apparently of limited physiological significance in domes- tic ruminants. In sheep, cortisol binding to Specific globular protein is low relative to non-ruminant species (Linder, 1964). Synthetic glucocorticoids have been used success- fully (Adams and Wagner, 1970) to induce fetal expulsion in sheep and cattle, i.e. a single 20 mg injection of 9-a F Prednisolone within a range of 250-293 days was ef— fective. More recently Jochle gE_al. (1971) induced par- turition in the bovine with a single 10 mg intramuscular 13 injection of Flumethasone at day 270 of pregnancy and 100 mg progesterone daily could block this effect. Both inves- tigators reported a high incidence of dystosia and retained fetal membranes. Prolactin Pituitary prolactin concentration remains relatively unchanged throughout pregnancy then increases markedly near parturition in rats (Reece EE.E£°' 1939), rabbits and guinea-pigs (Holst and Turner, 1939) and mice (Hurst and Turner, 1942). The increase of prolactin occurs within hours before or after delivery of young in all cases. This pattern established with bioassay of pituitary prolactin has recently been confirmed by radioimmunoassay of serum prolactin. Amenomori SE.E£' (1970) reported prolactin con- centration to average 8.3 ng/ml during pregnancy, increased to 29.2 ng/ml one day prepartum and peaked at 65.5 ng/ml on the day of parturition. Thereafter prolactin was maintained at 50 ng/ml presumably by the suckling stimulus. Everett and Baker (1945) observed a constant pituitary acidophil pOpulation in rats during pregnancy which increased 100% by the third day postpartum. Schams and Karg (1970) using radioimmunoassay de- tected a rapid increase in plasma prolactin beginning 12 hours prepartum and continuing through day 2 in the bovine. Peak plasma prolactin concentration at parturition exceeded 300 ng/ml. But, average serum prolactin values during late 14 pregnancy (dry period) and lactation were not significantly different. These results have been confirmed by Arije and Wiltbank (1971) with beef cows. Plasma prolactin measured at 90, 180 and 260 days of pregnancy in dairy cows averaged 220, 145 and 365 ng/ml, reSpectively (Oxender, 1971) but differences between these values were not significant. The physiological significance of increased pitui- tary and plasma prolactin at parturition is equivocal. A possible role in the multiplicity of endocrine events which culminate in initiation of lactation has been postulated by Meites (1966). This hypothesis holds that: (a) during pregnancy there is insufficient prolactin and adrenocortical hormones or both to initiate lactation. Both hormones are required to initiate lactation in the guinea pig (Folley, 1956), rat (Lyons eE_al., 1958) and goat (Cowie gt 31., 1964b); (b) estrogens and proqestins, which are secreted in large quantities during pregnancy prevent the stimulatory action of prolactin and corticoids on the mammary cells; and (c) at the time of parturition, prolactin and corti- coids increase concurrently with a decline in estrogen and progesterone resulting in the onset of lactation during pregnancy. Because multiple stimuli will evoke prolactin release in ruminants (Bryant SE 31., 1970; Tucker, 1971 and Raud g£_al., 1970) the possibility that increased serum prolactin at parturition may be a non-specific response to massive stimuli associated with parturition must be con- sidered. 15 During early lactation (day 1-8) serum prolactin concentration remains elevated in rats actively nursing litters (Amenomori gt’al., 1971). In contrast, average serum prolactin concentration during early lactation in cows is not different than during pregnancy (Schams and Karg, 1970; Arije and Wiltbank, 1971) but prolactin in- creases in cows and goats in response to milking stimuli (Bryant, Linzell and Greenwood, 1970; Tucker, 1971; and Raud 35 ii" 1971). The difference in reSponse of rats and large animals may reflect differences in suckling or milking frequency. Growth Hormone There is a puacity of information concerning serum and pituitary growth hormone (GH) concentration during gestation and parturition and the physiological role of GH at this time is equivocal. Bassett EE.§£° (1970) observed a prepartum increase in GH in three ewes, but the time of increase was variable ranging from 10-1 days prepartum. Oxender (1971) reported serum GH concentration to be 6,8 and 10 ng/ml at 90, 180 and 260 days gestation in cows, but differences between means were not significant. Similarly, Grumbach 35 El. (1968) reported pregnancy concentration of serum GH to average 7.0 ng/ml in women with no apparent fluctuations during gestation. Trenkle (1970) working with beef cattle found that feeding diethylstilbestrol at 10 mg per day significantly 16 increased plasma GH. Increasing serum estroqen near term (Hunter 33 al., 1970; Smith et_al., 1972) may stimulate GH release from the pituitary of cattle nearing parturition. The paucity of information on the role of GH during pregnancy and lactation probably reflects the fact that GH has not been reported to be a limiting factor to normal gestation. Yet the well documented role of GH in protein, carbohydrate and lipid metabolism (Evans 35 31., 1966) favors the view that this hormone, although not limiting, importantly contributes to normalcy of pregnancy. An adequate supply of GH is required for normal mammary growth and lactation (Meites, 1966). A lactational requirement for GH was established by Lyons (1958) and Lyons 35 31. (1958) using hypOphysectomized, gonadectomized and adrenalectomized rats. They determined that GH along with estrogen was required for mammary duct growth whereas GH plus estroqen, progesterone and prolactin was required for lobuloalveolar growth. Results obtained with mice (Elias, 1957; Rivera, 1967) or guinea pig (Gemtsen, 1960) mammary explants in 325£2_confirmed results obtained in After parturition, GB is required to obtain maxi- mum milk production. Cowie £5 31. (1964) reported that GH could increase and maintain milk production in hypOphysec- tomized goats when administered with prolactin, insulin, glucocorticoids and thyroid hormone. The important l7 contribution of GH in increasing lactation performances in these goats was shown by an immediate drop in milk produc- tion following GH withdrawal even though therapy with the other hormone was continued. Wrenn and Sykes (1953) reported GH administration markedly improved milk production in heifers in which lac- tation had been induced by estrOgen and progesterone therapy. The increase in milk production was much greater than that obtained with prolactin or crude pituitary ex- tract. In reviewing the work concerning the galactopoetic effect of GH, Meites (1961) cited the data of Shaw (1955) and Chung (1955) indicating GH administration for 9 days prepartum until 16 days after calving resulted in increased production during the entire lactation period. Brumby (1956) as cited by Meites (1961) was unable to confirm these observations but found an increase until day 7 post— treatment. Luteinizing Hormone Limited information is available concerning the LH pattern during pregnancy and around parturition. Labhset- war 35 31. (1964) demonstrated pituitary LH concentration to be lower at parturition than at days 260 or 265 of preg— nancy or day 21 postpartum. Using radioimmunological tech- niques Randel and Erb (1971) reported that LH decreased significantly from day 0 to day 7 of pregnancy and changed 18 very little thereafter. Using serum LH values in a multi- ple regression equation for predicting progesterone con- centration indicated a negative partial regression for LH which they interpreted to mean that LH may exert tonic regulation of luteal function during pregnancy. Postpartum Period Reproductive Tract Changes Uterus and Cervix.--The economic merits of a short calving interval has Spirited a great deal of research on the postpartum period. Palpation via the rectum has al- lowed researchers to monitor changes in uterine horn and cervical diameter as well as ovarian activity. According to Morrow (1969), the uterine horns of cattle are palpable by 4 to 7 days postpartum. A slow decrease in diameter was witnessed from days 4 to 9 followed by a more rapid decrease during days 10 to 14. The period of greatest involution occurred at the time of first estrus in normal cows and was concomitant with uterine lochia discharge. Cows with abnormal parturitions required 3 to 5 days longer to attain uterine size comparable to normal cows. Cervi- cal involution appeared to continue gradually until day 30 in normal cows and day 35 in problem animals after which time no further decrease was discernible by palpation. Wagner and Hansel (1970) have reported similar findings. They also indicated that in most normal cows the uterine mucosal epithelium was reestablished by 30 days postpartum. 19 In sheep uterine involution is completed by about 30 days postpartum (Uren, 1935; Basset, 1963 as cited by Wagner and Oxenreider, 1971). Ovarian Activity Casida and Venzke (1936) and Labhsetwar (1964) indicated mature follicles were present around 30 days postpartum in cattle. According to Morrow gt El' (1969), ovarian follicular activity commences 7 to 10 days post— partum at which time follicles averaged 0.5 to 1.5 cm in diameter. Enlargement of such follicles continued until first estrus at approximately 15 days. The corpus luteum of pregnancy had regressed to a small elevated mass on the ovarian surface at 4 to 7 days postpartum and by 14 days it was usually undetectable. Postpartum Estrus Activity Morrow et 31. (1966) reported the interval from calving to first postpartum estrus to be 15.0 days in normal cows and 34.4 days for abnormal cows. Abnormal cows in this study were animals that experienced dystocia, ketosis or other disease conditions shortly after parturi- tion. A 14.0 day interval was reported by Wagner and Hansel (1969). Other reports have indicated that the first postpartum ovulation occurred somewhat later (Saiduddin gt 31., 1967a; Tennant gt 31., 1967). In most reports indicating an interval longer than 15.0 days 20 palpations were initiated to detect an early ovulation. Morrow st 31. (1966) found 79% of the first ovulations were accompanied by silent heats (ovulation without clini- cal signs of estrus). Menge st 31. (1962) and Saiduddin gt al. (1967b) have reported similar observations. In comparing data on this subject, many of the discrepancies witnessed are dependent on the quality and thoroughness of estrus detection and estrus classification criteria. First postpartum estrus cycle length (first ovula- tion to second ovulation) was 16 to 17 days in normal and 19.7 days in abnormal cows compared to a second cycle length of 21 days (Morrow gt 31., 1966; Marion and Gier, 1967). Morrow EE.E£° (1969) have Speculated that the short cycle is due to failure of the corpus luteum earlier than usual. Oxytocin injections, uterine dilation and intrauterine infusions of contaminated seminal plasma early in the cycle all cause similar reductions in cycle length (Hansel and Wagner, 1960). This implicates the uterus in corpus luteum maintenance, perhaps by decreasing LH production by the anterior pituitary. Wagner and Oxenreider (1970) reviewed the data from several other Species. Horses apparently exhibit an ovulatory estrus within 18 days of foaling and pigs have an anovulatory estrus l to 3 days postpartum. Postpartum suckling and low energy diets appear to be two factors which can increase the postpartum 21 interval to first estrus. Both may exert their influence via disturbed hypothalamic control of gonadotropin secre— tion. McClure (1968a,b) postulated poor nutrition might result in hypoglycemia and this in turn might disturb hypo- thalamic control of the anterior pituitary. Endocrine Pattern The endocrine profile during this time has been very poorly defined. Most work has been limited to pituitary or ovarian levels of hormones but blood levels are now measur- able with radioimmunoassay procedures. Luteinizing hormone concentrations of bovine pitui- taries increase from parturition to day 21 postpartum and to first estrus (Labhsetwar gt_al., 1963; Saiduddin gt 31., 1964). Saiduddin st 31. (1966) showed pituitary LH activity to be lowest at parturition, increased to day 10 and then continued a gradual increase until day 30. Pituitary FSH levels were highest at parturition and shortly after but then declined somewhat. In humans, plasma FSH postpartum was quite constant from parturition until day 30 but these levels were about one-half of those found during the folli- cular phase of the cycle (Crystle SE 31., 1970). Luteiniz- ing hormone activity was highest near parturition and de- creased thereafter but the assay procedure used cross reacted with human chorionic gonadotropin (HCG) thus ex- plaining the high levels of LH activity during the early postpartum period. MATERIALS AND METHODS Experimental Design This thesis was designed to quantify changes in blood hormone concentrations of the bovine during the last month of gestation and the interval between calving and first estrus. Jugular blood (40 ml) was collected via venipunc- ture from each of 34 pregnant Holstein heifers twice weekly from 30 to 6 days prepartum, twice daily (8:00 AM and 5:00 PM) from 6 days before to 5 days after parturition then twice weekly until first estrus or day 25 postpartum whichever occurred first. All animals were in loose housing with access to pasture ad libidum, supplemented with corn silage, hay and grain concentrate. Animals were placed in individual maternity pens (when prepartum twice daily bleedings were started) where they remained until 48 hours post-calving. Estrus Detection Following parturition, the heifers were observed twice daily for signs of estrus. In addition, beginning 1 week postpartum twice weekly rectal palpations were 22 23 initiated to monitor ovarian activity and changes in repro- ductive tract size. This allowed us to detect both animals diSplaying signs of estrus and those with silent heats. Blood Handling Procedure Blood was placed in 50 m1 polypropylene centrifuge tubes (Ivan Sorvall, Inc., Norwalk, Conn.) containing 31.7 mg oxalic acid crystals, centrifuged (6500 xg, 20 min. 4°C) and plasma transferred to similar centrifuge tubes contain- ing 27.8 mg CaCl2 to promote clot formation. After 48 hours at 5°C, samples were centrifuged as before to remove the fibrin clots and serum was transferred to 7-dram plastic vials and stored at -20°C until assayed for hormones. Radioimmunoassays A double antibody radioimmunoassay (RIA) was em- ployed to quantify serum LH, GH and prolactin. The proced- ures for these assays were similar to those reported by Niswender st 31. (1969) for bovine LH. Descriptions of the prolactin (Tucker, 1971 and Koprowski and Tucker, 1971) and GH (Purchas, 1970) and LH (Swanson, 1970) assay methodology has been previously described. General Principles of Radioimmunoassay As opposed to steroid hormones which possess dis- tinct chemical groupings and solubility characteristics allowing separation from other plasma components, protein 24 or peptide hormones are much less distinguishable and not easily isolated from other plasma proteins. These hormones are however antigenic and this phenomena has allowed devel- opment of immunoassays which are sensitive and precise even in the presence of a multiplicity of proteins. The production of an antiserum specific for one hormone is the first phase of the RIA procedure. This antibody must combine specifically with the hormone antigen to produce an antigen-antibody complex. Displacement of a radioactively labeled antigen (hormone) by unlabeled or "cold" antigen in proportion to its concentration is used to quantify hormone concentrations in unknowns by compari- son with the displacing capacities of standard amounts of unlabeled hormone. Finally, by equating unknown values to standard values the quantity of unknown can be cal- culated. Antibodies Antibodies against LH, GH and prolactin were in- duced in guinea pigs by repeated injections of purified NIH preparations of these hormones in Freunds adjuvant (Appen- dix I.C.l). Such antibodies are referred to as the first antibody. According to Yalow and Berson (1968) maximum assay sensitivity is obtained when approximately 33% of the labeled hormone is bound by the antibody preparation. This was generally obtained when first antibody was diluted l:200,000 for LH; 1:3,200 for GH and l:30,000 for prolactin. 25 The second antibody was a sheep or goat preparation of anti—guinea pig gamma globulin obtained by a method similar to that used to induce the first antibody (Appen- dix I. C.2). The purpose of the second antibody was to precipitate the first antibody-hormone complex and was used at a dilution to yield maximum precipitation, i.e., usually 1:4 - 1:6. Labeled Hormone Preparation Mixing a purified preparation of the protein hor- mone (5 ug of GH and prolactin; 2.5 ug LH) with approxi- 1251 (50 mc/ml, Iso-Serve Division of Cam- mately 1 mo of bridge Nuclear Corporation, Cambridge, Mass.) in the pre- sence of chloramine-T resulted in labeling of the ortho positions of tyrosyl residues (Appendix I. A.3). Reaction time was confined to 2 minutes and was stOpped by adding sodium metabisulfate (Appendix I. A.4). A transfer solu- tion (Appendix I. A.5) was added to the reaction mixture and it was layered on top of a column of Bio-Gel-P-60 (Bio-Rad Labs., Richmond, Calif.) (Appendix I. 3.2) and 15 1.0 ml fractions were collected in 12 x 75 mm disposable glass culture tubes containing 1 ml egg white albumin in phosphate buffered saline (2% EWA-PBS) for LH and bovine serum albumin in phosphate buffered saline (2% BSA-PBS) for GH and prolactin. This column allowed separation of the labeled hormone from the free iodine. A typical elu- tion profile using LH as the example is represented in 26 Figure 1. The labeled hormone was diluted to yield a solu- tion containing approximately 25,000 cpm per 100 ul. The diluent for LH was 1% EWA-PBS (Appendix I. 8.3) and 1% BSA-PBS for CH and prolactin (Appendix I. 8.3). Radioimmunoassay Each serum sample was assayed in duplicate. Growth hormone and LH concentrations were estimated in undiluted serum aliquots whereas serum was diluted 1:2 to 1:10 with 1% bovine serum albumen in BSA-PBS for prolactin quantita— tion. The serum sample volume used ranged from 300 to 500 ul. Samples were added to 12 x 75 mm disposable glass tubes containing either 1% eggwwhite albumin in phosphate buffered saline (LH) or 1% BSA-PBS for GH and prolactin so that the final volume of serum plus diluent constituted 500 ul. Four sets of standards (Appendix I. B.4) were evenly distributed throughout the assay and each tube con- tained 500 ul of the standard. Table 1 illustrates the mechanics of the radioimmunoassay procedure. The radioactivity of the precipitate was determined by counting in an automatic gamma counter for 10 minutes or 10,000 counts whichever accumulated first. Unknown samples were quantified by comparing with the standard. 27 300 r- ? 240_ 00 C) T epm/ml no no (3 CD CD I . ' . 1 2 4 6 8 l0 l2 l4 ml of effluent 9 p Figure 1.--Radioactivity elution profile for Bio- Gel P-60 chromatography of iodinated luteinizing hormone (LH). The first peak represents iodinated LH and the second peak represents free iodine. 28 .wcfiamm cmuowmsn wumnmmonm m .cwasnon mssmm mam mocfism «new mmmnmv .ocosnon npzoum mcfl>onnfiucm mam mmcwsom .cauomaoum mca>onlwucm mam «meadow .mcosuon msfluacwmuda mcfi>onnwpcm mam mmcwsUH SDMMm mqomz as ooa m24m oov memmpqu H5 com a: ooa as com Qz¢ wEomm mo Imomdmo as com me mNH m oaua I mug ,umBDqHQ ZDmHm as ooa mzdw uov mammoqu Ha ooa as ooa a: ooa 02¢ 98% mmadgm Mania 3 oou 229305 .938 97.500 .mmmPH. gmmm mqomz use .ezcemzmmnpm mmo moon as ooa wxoamm ad sz on muamHmBsz oov madeUzH as com a: ooa as com Q24 as m IoEmEm mg Imqmgmo as com 5 v mad H MaomHBz¢ mzozmom woomHBz¢ Gmoad ZDme omaad mmmm azoumm QHBGZHQOH BmMHh mo QmBDAHQ m0 QAOU mo MEDAO> ho NEDAQ> mo NSDQO> WZDQO> 3mm m0 .qo> m ~40 m Mme v wen m >¢Q N >4a H wen wan mp 9mm .muapwuoum wmmmmossssfloHpmm|n.H manna RESULTS Pre and Postpartum Serum Prolactin, GH and LH Concentration in Heifers Serum Prolactin Serum prolactin concentration of heifers from 26 days prepartum through 26 days postpartum are shown in Figure 2 excluding details of the period from prepartum day 9 through postpartum day 9 which are shown on Figure 3. From day 26 to 2 days before calving, prolactin concentra- tions varied between 80 and 110 ng/ml. But, from day 2, serum prolactin concentration increased rapidly reaching a peak of 285 ng/ml 1 day before calving with a gradual de- cline thereafter. Serum concentrations were 217 ng/ml at parturition and 93 ng/ml on postpartum day 2. From day 2 until day 9 serum prolactin values fluctuated between 93 and 86 ng/ml (Figure 2). Following day 9 a gradual de- crease continued until day 26 when the concentration was 36 ng/ml. Postpartum concentrations (Figure 2) were generally lower than those found during the final month of gestation. In contrast to GH which peaks at parturition, prolactin values were maximum at 24 hours prior to calving and de- clined thereafter. 29 30 zo_._._m3._.mm© m scum awuomaoum Enummnu.m musmwm 295553 5.52 mo mmonmm 92a mini??? _. o _- m- Tim- ~\1‘ _ q q 4 . d . eiq . a J . . q . d . . .7. MOFIJI.AV L .. s , 3 . L m. L00. W .. d 1 U. , O . .I l V . m .OON M . m: .D L . / m. zo_._..mD.r¢mw mm Eoum wcosnog Busouo Esummun.v musoflm ZO_._._mD._.m